Peaceful coexistence with the essential microbes that populate the mammalian gut requires a careful balance between tolerance of commensals, maintenance of barrier function, and avoidance of damaging inflammation. A key role is played by FoxP3+ T regulatory (Treg) cells, which help maintain immunologic tolerance and control inflammation in many organismal contexts. We have recently described the induction by several symbionts from the human gut of a unique population of FoxP3+ Treg cells that also express and require functionally the transcription factor RORg. This is paradoxal, as RORg is the master regulator of pro- inflammatory Th17 cells. Rorg+ Treg cells expand in response to gut symbionts, perhaps through local differentiation, and are functionally involved in the control of intestinal inflammation. They co-exist in the colon with more typical Helios+Gata3hi Tregs, likely of thymic origin. We propose to address several important questions opened by these observations, to explore the control, origin and function of Rorg+ Tregs. 1. How do FoxP3 and RORg synergize at the molecular level, and how does RORg function so differently in Th17 cells vs Rorg+ Tregs? This will be addressed by analyzing transcription and chromatin changes resulting from carefully controlled expression of FoxP3 and RORg in CD4+ T cells, modulated with other transcription factors that are differentially represented in Th17 and Tregs, or with oxysterol ligands of RORg. These inferences will be validated in vivo with conditional knockout mice, and their relevance to human physiology will be assessed by transducing human CD4+ T cells in parallel, and by low-input RNAseq analysis of Rorg+ Tregs we found in the human colon. 2. What are the cellular origin and differentiation pathways of Rorg+ Tregs? The origin and inter- relationships between colonic Treg subsets in the colon will be analyzed by transfer experiments, by lineage tracing after Treg-specific tagging, and by using TCR sequences as barcodes to assess relationships between Treg and Tconv populations in mice colonized by a single microbe 3. Relative roles of RORg+ and Helios+ colonic Tregs assessed by inducing Treg-specific knockouts in the key transcription that reciprocally control colonic Treg populations (Rorc, Gata3, Ikzf2). The effects of these perturbations will be assessed on colon inflammation at baseline, the control of chemical or bacterially- induced inflammation, intestinal barrier integrity, microbe-specific IgA repertoire and whether changes in Treg populations influence the balance of microbial phyla and species. These connected explorations will provide a unique mechanistic and functional understanding of these essential Treg populations, and will have profound implications on our understanding of the control of inflammation at the host/symbiont interface.